The quantification of chemical and biochemical components in colored aqueous fluids, in particular, colored biological fluids such as whole blood and urine and biological fluid derivatives such as serum and plasma, is of ever-increasing importance. Important applications exist in medical diagnosis and treatment and in the quantification of exposure to therapeutic drugs, intoxicants, hazardous chemicals, and the like. In some instances, the amounts of materials being determined are either so minuscule--in the range of a milligram or less per deciliter--or so difficult to precisely determine that the apparatus employed is complicated and useful only to skilled laboratory personnel. In this case, the results are generally not available for some hours or days after sampling. In other instances, there is often an emphasis on the ability of lay operators to perform the test routinely, quickly, and reproducibly outside a laboratory setting with rapid or immediate information display.
One common medical test is the measurement of blood glucose levels of diabetics. Current teaching counsels diabetic patients to measure their blood glucose level from two to seven times a day, depending on the nature and severity of their particular cases. Based on the observed pattern in the measured glucose levels, the patient and physician together make adjustments in diet, exercise, and insulin intake to better manage the disease. Clearly, this information should be available to the patient immediately.
Many blood glucose test methods and test articles are known in the art; these all suffer from a variety of limitations. A great improvement is disclosed and claimed in U.S. Pat. Nos. 4,935,346, 5,049,487, 5,059,394 and 5,179,005 by R. Phillips et al. and as assigned to the same assignee as the present application.
The method disclosed and claimed in these patents involves taking a reflectance reading from one surface of an inert porous matrix impregnated with a reagent that will interact with the analyte to produce a light-absorbing reaction product when the fluid being analyzed is applied to another surface and migrates through the matrix to the surface being read. The reagent includes glucose oxidase, an enzyme which consumes glucose in the sample to produce hydrogen peroxide which, in the presence of another enzyme, horseradish peroxidase, oxidizes a dye couple comprising 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) and 3-dimethylaminobenzoic acid (DMAB) to yield a blue dye. Reflectance measurements are then made at two separate wavelengths. The concentration of the glucose in blood is determined based on the intensity of the dye color with the aid of a LED spectrophotometer.
In my commonly assigned U.S. Ser. No. 245,940, filed May 19, 1994, now U.S. Pat. No. 5,453,360, (LFS 30), there is disclosed a dye couple comprising 3-methyl-2-benzothiazolinone hydrazone in free form or in acid form (MBTH) and 8-anilino-1-napthalenesulfonate, in acid or salt form (ANS) to be used in place of the MBTH-DMAB dye couple as described above. The MBTH-ANS dye couple is less soluble upon oxidation and, hence, provides a more stable endpoint, with minimal dye fading, as compared to the oxidized MBTH-DMAB dye couple.
While these prior systems have been effectively employed to produce useful test devices for the determination of the presence or quantity of glucose, several drawbacks have been noted. The test devices in which such dye couples are employed are designed for both home use and for professional use and as such are sold by the manufacturers and distributors with the expectations that they will remain in the inventory of the user for a substantial period of time and must, of course, remain effective over this period of time. This need for a substantial shelf life has caused difficulties in the formulation of products employing MBTH as one of the components of a dye couple.
Firstly, it has been found that the stability of MBTH decreases with increasing temperature and alkalinity. The acid free form of MBTH is very labile and tends to sublime away. In an attempt to counter this, a preferred form is the acid hydrate of MBTH e.g., 3-methyl-2-benzothiazolinone hydrazone hydrochloride. Unfortunately, this hydrate is itself instable upon increasing temperature and readily dissociates into acid free MBTH and HCl upon heating. In additional to having low stability at high pH, the efficiency of MBTH to oxidatively react with its coupling partner greatly decreases with increasing alkalinities so that at high pH essentially little or no color is produced from the dye couple.
In view of these relationships, in practice MBTH must be used in large excesses and at low pH to minimize the effects of instability and inefficiency. Ideally, a pH of below 2.0 would be preferred from the point of view of low sublimation and high efficiency of the compound. Unfortunately, for the systems being considered herein such an ideal low pH cannot be employed. As described above, the reagent systems employed depend upon enzymes to act on the substrate analyte and generate oxidation agents in quantities indicative of the quantities of the analyte present in the sample being tested. The low pH, which would be ideal with respect to the MBTH reagent, is entirely unsuitable for enzymes such as, for example, glucose oxidase and horseradish peroxidase. At such low pHs many of such commercially available enzymes have little or no activity. Accordingly, the art has been forced to comprise and chose a moderate pH e.g., 4, and a great excess of reagents to insure efficiency of their test devices for the required shelf life.